Excessive HSP70/TLR2 activation leads to remodeling of the tumor immune microenvironment to resist chemotherapy sensitivity of mFOLFOX in colorectal cancer.

For locally advanced colorectal cancer (CRC), neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision or complete mesocolic excision is the standard therapeutic strategy, which is key to patient survival. Involvement of the tumor immune microenvironment is a factor that regulates tumor progression and sensitivity to nCRT in CRC. In this study, we aimed to identify the effect of heat-shock protein 70 (HSP70)/toll-like receptor-2 (TLR-2) on mFOLFOX sensitization for CRC. A total of 22 patients with advanced CRC who had received neoadjuvant mFOLFOX were enrolled and classified into the mFOLFOX-insensitive or -sensitive group, according to the tumor regression grade. The abundance of immune infiltrates was significantly higher in the post-operative pathological specimens of the mFOLFOX-insensitive group, as compared to those of the mFOLFOX-sensitive group. After transcriptome sequencing, differentially expressed genes between the two groups were annotated to inflammatory and immune responses using Gene Ontology (GO) analysis, and the TLR signaling pathway was analyzed using Kyoto Encyclopedia of Genes and Genomes pathway analysis. Significantly higher expression levels of HSP60, HSP70, HSP90, and TLR-2 in the mFOLFOX-insensitive group were detected using immunofluorescence assays. TIMER2.0 platform was introduced to further narrow the scope of HSP70 (HSPA6 or HSPA7) and TLR-2, which exhibited positive correlations with dendritic cells, Tregs, or CD4+ T cells and negative correlations with CD3+ or CD8+ T cells, implying that HSP70/TLR-2 activation mediates immunosuppressive cells to counteract CD8+ T cells, which may be a novel target of CRC treatment. A promising synergistic effect of mFOLFOX combined with a TLR-2 inhibitor was observed in vivo in mouse allograft models, which could be partly rescued by recombinant HSP70 protein. Immunohistochemical staining of allografts and immunofluorescence assays of clinical specimens corroborated the regulatory effects of the immune microenvironment. In summary, HSP70/TLR-2 activation can regulate the tumor immune microenvironment of CRC and further remodel its sensitivity to mFOLFOX. However, the specific mechanisms remain unclear and require further investigation. This study is expected to provide a new direction for the clinical treatment of CRC.

Oncologic outcomes of single-incision laparoscopic surgery versus conventional laparoscopic surgery for colorectal cancer (CSILS): study protocol for a multicentre, prospective, open-label, noninferiority, randomized controlled trial.

BACKGROUND: In most previous studies, single-incision laparoscopic surgery (SILS) for colorectal cancer (CRC) was feasible and safe in the short term. However, long-term oncologic outcomes remain uncertain, as only a few studies contained long-term survival data. SILS for CRC is still in the early stages of research. Further studies, particularly large-scale, prospective randomized controlled trials, are necessary to assess the value of SILS for CRC. METHODS: This study is a prospective, multicentre, open-label, noninferiority, parallel-group randomized controlled trial that investigates the long-term oncologic outcomes of SILS compared to conventional laparoscopic surgery (CLS) for CRC. A total of 710 eligible patients will be randomly assigned to the SILS group or the CLS group at a 1:1 ratio using a central, dynamic, and stratified block randomization method. Patients with ages ranging from 18 to 85 years old, of both sexes, with CRC above the peritoneal reflection diagnosed as cT1-4aN0-2M0 and a tumour size no larger than 5 cm will be considered for the study. The primary endpoint is 3-year disease-free survival (DFS). The secondary endpoints include: intraoperative outcomes, postoperative recovery, postoperative pain assessment, pathological outcomes, early morbidity and mortality rate, cosmetic effects, quality of life, 3-year overall survival (OS), incidence of incisional hernia, 5-year DFS and 5-year OS. The first two follow-up visits will be scheduled at one month and three months postoperatively, then every three months for the first two years and every six months for the next three years. DISCUSSION: Currently, no randomized controlled trials (RCTs) have been designed to investigate the long-term oncologic outcomes of SILS for CRC. This study is expected to provide clinical evidence of the oncologic outcomes of SILS compared to CLS for CRC to promote its widespread use. TRIAL REGISTRATION: ClinicalTrials.gov:  NCT04527861 (registered on August 27, 2020).

Short-Term Outcomes of Single-Incision Laparoscopic Surgery for Colorectal Cancer: A Single-Center, Open-Label, Non-Inferiority, Randomized Clinical Trial.

OBJECTIVE: To date, well-designed randomized controlled trials examining the safety, efficacy, and long-term outcomes of single-incision laparoscopic surgery (SILS) for colorectal cancer are scarce. The aim of the current study was to compare short-term outcomes of SILS for colorectal cancer with conventional laparoscopic surgery (CLS). METHODS: Between June 28, 2017, and June 29, 2019, a single-center, open-label, non-inferiority, randomized clinical trial was conducted at the Department of General Surgery, Ruijin Hospital (North), Shanghai Jiaotong University School of Medicine in Shanghai, China. In total, 200 patients diagnosed or suspected of colorectal cancer (cT1-4aN0-2M0) were randomly assigned to either the SILS or CLS group in a 1:1 ratio. The primary outcome was early morbidity rate. Secondary outcomes included intraoperative outcomes, pain intensity, postoperative recovery, pathologic outcomes, and long-term outcomes. RESULTS: In total, 193 participants (SILS, 97; CLS, 96) were analyzed in the modified intention-to-treat (MITT) population. Among them, 48 underwent right hemicolectomy (SILS n = 23, 23.7% and MLS n = 25, 26%), 15 underwent left hemicolectomy (SILS n = 6, 6.2% and MLS n = 9, 9.4%), 1 underwent transverse colectomy (MLS n = 1, 1%), 57 underwent sigmoidectomy (SILS n = 32, 33% and MLS n = 25, 26%), and 72 underwent anterior resection (SILS n = 36, 37.1% and MLS n = 36, 37.5%). No significant differences were observed in the baseline characteristics. The intraoperative complication was comparable between the two groups [5 (5.2%) vs. 4 (4.2%); difference, 1%; 95% CI, -5.8% to 7.8%; p > 0.999) and so was postoperative complication rates [10 (10.3%) vs. 14 (14.6%); difference, -4.3%; 95% CI, -13.9% to 5.3%; p = 0.392]. The SILS group showed shorter incision length [median (IQR), 4 (3.5-5) vs. 6.6 (6-7.5), p < 0.001] and lower VAS scores on the first [median (IQR), 4 (3-5) vs. 4 (4-5), p = 0.002] and the second day [median (IQR), 2 (1.5-3) vs. 3 (2-4), p < 0.001] after surgery. No statistically significant difference was found in other measured outcomes. CONCLUSIONS: Compared with CLS, SILS performed by experienced surgeons for selected colorectal cancer patients is non-inferior with good short-term safety and has the advantage of reducing postoperative pain. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT03151733.

Comparative Effectiveness of Enhanced Recovery After Surgery Program Combined With Single-Incision Laparoscopic Surgery in Colorectal Cancer Surgery: A Retrospective Analysis.

BACKGROUND: Recently, enhanced recovery after surgery (ERAS) has been widely used in the perioperative management of colorectal cancer (CRC). This study aimed to evaluate the safety and feasibility of ERAS combined with single-incision laparoscopic surgery (SILS) in CRC surgery. METHODS: This was a retrospective study of patients with CRC who underwent surgery between April 2018 and April 2020 in Ruijin Hospital(North), Shanghai Jiaotong University School of Medicine. The patients were divided into three groups: group A (n=138), patients who underwent traditional multiport laparoscopic colectomy with conventional perioperative management; group B (n=63), patients who underwent SILS; and group C (n=51), patients who underwent SILS with ERAS. RESULTS: Overall, 252 participants were included in the retrospective study. The median operation time (min) in group B and group C was shorter than that in group A (group A 134.0 ± 42.5; group B 117 ± 38.9; group C 111.7 ± 35.4, p=0.004). The estimated surgical blood loss (ml) was lower in groups B and C than in group A (group A 165.1 ± 142.2; group B 122.0 ± 79.4; group C 105.2 ± 55.8, p=0.011). The length of surgical incision (cm) was shorter in groups B and C than in group A (group A 7.34 ± 1.05; group B 5.60 ± 0.80; group C 5.28 ± 0.52, p<0.001). The time before first flatus (hours) in group C was shorter than in groups A and B (group A 61.85 ± 21.14; group B 58.30 ± 20.08; group C 42.06 ± 23.72; p<0.001). The days prior to the administration of free oral fluids in group C was shorter than in groups A and B (group A 4.79 ± 1.28; group B 4.67 ± 1.11; group C 2.62 ± 0.64; p<0.001). The days of prior solid diet was less in group C than in groups A and B (group A 7.22 ± 3.87; group B 7.08 ± 3.18; group C 5.75 ± 1.70; p=0.027). The postoperative length of stay (LOS) was less in group C compared with that in groups A and B (group A 9.46 ± 4.84 days; group B 9.52 ± 7.45 days; group C 7.20 ± 2.37 days; p=0.023). The visual analog scale (VAS) scores on day 0, 1, and 2 in groups B and C were lower than those in group A (day 0, p<0.001; day 1, p<0.001; day 2, p=0.002), while the VAS score on day 3 showed no differences in the three groups (group A 1.29 ± 1.38; group B 0.98 ± 1.24; group C 0.75 ± 0.64, p=0.018). CONCLUSION: The findings suggest that SILS combined with ERAS may be a feasible and safe procedure for CRC surgery because it provides favorable cosmetic results, early dietary resumption, shorter hospital stays, and appropriate control of postoperative pain without increases in complications or readmission rates compared to conventional perioperative care with SILS or conventional laparoscopic surgery(CLS) of CRC. Further prospective randomized controlled studies are needed to enhance evidence-based medical evidence.

Cell-Cycle-Dependent Phosphorylation of PRPS1 Fuels Nucleotide Synthesis and Promotes Tumorigenesis.

Nucleotide supply is essential for DNA replication in proliferating cells, including cancer cells. Ribose-phosphate diphosphokinase 1 (PRPS1) is a key enzyme to produce the consensus precursor of nucleotide synthesis. PRPS1 participates in the pentose phosphate pathway (PPP) by catalyzing the phosphoribosylation of D-ribose 5-phosphate (R-5P) to 5-phosphoribosyl-1-pyrophosphate. Therefore, PRPS1 not only controls purine biosynthesis and supplies precursors for DNA and RNA biosynthesis but also regulates PPP through a feedback loop of the PRPS1 substrate R-5P. However, it is still elusive whether PRPS1 enhances nucleotide synthesis during cell-cycle progression. In this study, we explore the role and activation mechanism of PRPS1 in cell-cycle progression of colorectal cancer, and observed a peak in its enzymatic activity during S phase. CDK1 contributes to upregulation of PRPS1 activity by phosphorylating PRPS1 at S103; loss of phosphorylation at S103 delayed the cell cycle and decreased cell proliferation. PRPS1 activity in colorectal cancer samples is higher than in adjacent tissue, and the use of an antibody that specifically detects PRPS1 phosphorylation at S103 showed consistent results in 184 colorectal cancer tissues. In conclusion, compared with upregulation of PRPS1 expression levels, increased PRPS1 activity, which is marked by S103 phosphorylation, is more important in promoting tumorigenesis and is a promising diagnostic indicator for colorectal cancer. SIGNIFICANCE: These findings show that the enzymatic activity of PRPS1 is crucial for cell-cycle regulation and suggest PRPS1 phosphorylation at S103 as a direct therapeutic target and diagnostic biomarker for colorectal cancer.

The impact of cation acidity and alkyl substituents on the cation-anion interactions of 1-alkyl-2,3-dimethylimidazolium ionic liquids.

In this study, eight 1-alkyl-2,3-dimethylimidazolium ionic liquids are analysed by X-ray photoelectron spectroscopy. The effect of both the anion and the cation on the electronic environment of cationic nitrogen regions is explored. It concludes that the cationic N 1s binding energy shifts to the lower value when the basicity of the anion increases or the acidity of the cation decreases. The impact of the cation acidity on the cation-anion interactions is demonstrated systematically by carefully comparing the binding energies of anion-based components for each anion. It is found that for more basic anions, the charge-transfer effect between counterions can be effectively shielded; for less basic anions, such an effect is negligible. The charge shielding effect of the alkyl substituent is also studied by using dodecyl-based ionic liquids, compared to 1-alkyl-3-methylimidazolium analogues. It suggests that long alkyl substituents can have a significant electron donating effect to the cation headgroup and thus effectively shield the charge-transfer effect between cations and anions.

MLLT10 promotes tumor migration, invasion, and metastasis in human colorectal cancer.

OBJECTIVES: Colorectal cancer (CRC), one of the most aggressive gastrointestinal malignancies, is a frequently diagnosed life-threatening cancer worldwide. Most CRC patients have poor prognosis mainly because of frequent metastasis and recurrence. Thus, it is crucial to find out some new biomarkers and to show deeper insights into the mechanisms of CRC. MLLT10, Myeloid/lymphoid or mixed-lineage leukemia translocated to 10, also known as AF10, a recurrent MLL partner. In this study, we found that MLLT10 promotes CRC tumor invasion and metastasis both in vitro and in vivo. METHODS: Here, the expression of MLLT10 was evaluated by immunohistochemistry. Then, the plasmid and lentivirus particles for MLLT10 overexpression or knockdown were designed and constructed into SW620 and HT29 cells. Finally, cell proliferation assay, cell adhesion assay, transwell migration, and invasion assay were used to detect the migration and invasion ability of MLLT10 in CRC cells. A tail vein injection assay was employed to evaluate the role of MLLT10 in tumor metastases. RESULTS: MLLT10 expression was significantly higher in CRC tissues than in noncancerous tissues and was associated with some clinicopathological factors. In vitro, the overexpression of MLLT10 promoted CRC cell migration and invasion, while after MLLT10 was knocked down, the opposite results were observed. Furthermore, we used animal metastasis models to detect the function of MLLT10 in vivo, the results are same with the outcomes in vitro. In lung metastasis sites, the knockdown of MLLT10 in SW620 cells significantly inhibited Vimentin expression, whereas the E-Cadherin was increased. CONCLUSIONS: These results indicate that MLLT10 regulates the metastasis of CRC cells via EMT.

[Factors affecting the difficulty of laparoscopy-assisted triple-port anterior resection].

OBJECTIVE: To explore the factors affecting the operative difficulty of triple-port laparoscopic surgery (TLS) in anterior resection. METHODS: A retrospective case-control study was carried out. Clinical and MRI imaging data of 106 colorectal cancer cases undergoing TLS anterior resection at Department of Colorectal Surgery of Ruijin Hospital between 2013 and 2016 were retrospectively analyzed. INCLUSION CRITERIA: (1) patients receiving TLS anterior resection (Dixon operation); (2) preoperative stageI( to III( malignant tumor;(3) distance of 5-15 cm from inferior margin of tumor to anal verge; and (4) available preoperative rectal MRI. EXCLUSION CRITERIA: (1) patients receiving preoperative adjuvant therapy; (2) patients with low rectal cancer or with local advanced disease; (3) T4b tumor. Rectal MRI was introduced to measure the structure of pelvis. In sagittal view, superior margin of the first sacral vertebrae, superior margin of the third sacral vertebrae, apex of coccyx, and the line of superior margin of pubic symphysis were used to form a pentagon. The 5 lines were marked as N, O, P, Q, R, and the 5 included angles were marked as angle 1, 2, 3, 4, 5. Organs (uterus and prostate) and tumor (transverse diameter, longitudinal diameter, section area, lesion length, distance to circumference cutting edge) were also measured on MRI. The operative time was applied to be the indicator of operative difficulty and patients were divided into 2 groups according to median operative time. Baseline information (age, gender, BMI, distance from inferior margin of tumor to anal verge, operative history, length of tumor), preoperative tumor staging, and MRI measurements (pelvis, tumor, uterus, prostate), etc were compared between two groups. Factors affecting operative difficulty of TLS were analyzed with logistic regression model. RESULTS: Of 106 enrolled patients, 73 were male and 33 female with mean age of (59.8±12.2) years and mean BMI of (22.8±3.3) kg/m2; 25 patients had previous abdominal surgery; distance from inferior margin of tumor to anal verge was (7.4±2.0) cm and the tumor diameter was (3.7±1.4) cm; 24, 36 and 46 patients were in stage I(, II( and III( respectively. All operations were completed successfully. The median number of harvested lymph node was 13(11-16); the median length of distal resection margin was 2.5(2.0-3.1) cm; the median operative time was 2.0(1.5-2.6) hours; the median intraoperative blood loss was 50(0-100) ml; the median time to liquid diet was 4(3-5) days; the median hospital stay was 7(6-10) days. Ten cases (9.4%) developed complications within 30 days after surgery. Patients were divided into ≤2 h group and > 2 h group according to median operative time, and both groups had 53 patients. As compared to ≤2 h group, >2 h group had shorter distance from inferior margin of tumor to anal verge [(6.8 ± 1.5) cm vs. (8.0 ± 2.4) cm, t = 3.174, P = 0.004], lower ratio of (R+N)/(O+P)(1.61±0.27 vs. 1.73±0.19, t = 2.494, P = 0.014), larger transverse distance of tumor [(3.45±0.72) cm vs. (3.05±0.89) cm, t = 0.224, P = 0.027]. Multivariate logistic regression analysis showed the distance from inferior margin of tumor to anal verge was the independent factor affecting operative difficulty(OR=0.584, 95%CI:0.429-0.796, P = 0.001). CONCLUSIONS: Surgeons may have less difficulty in performing TLS anterior resection for patients with longer distance from inferior margin of tumor to anal verge. In preoperative assessment of operative difficulty of TLS, comprehensive evaluation should be performed. Distance from inferior margin of tumor to anal verge should be regarded as the main factor, and MRI (R+N)/(O+P) and transverse diameter of tumor should be used as important reference, leading to reasonable choice of cases for TLS and smooth pass of study curve.

Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer.

Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety in the treatment of advanced gastric carcinoma and some other solid tumors. However, the direct functional mechanisms of tumor lethality mediated by apatinib have not yet been fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, in this study, we demonstrated that apatinib could induce both apoptosis and autophagy in human colorectal cancer (CRC) via a mechanism that involved endoplasmic reticulum (ER) stress. Moreover, activation of the IRE1α pathway from apatinib-induced ER stress is responsible for the induction of autophagy; however, blocking autophagy could enhance the apoptosis in apatinib-treated human CRC cell lines. Furthermore, the combination of apatinib with autophagy inhibitor chloroquine (CQ) tends to have the most significant anti-tumor effect of CRC both in vitro and in vivo. Overall, our data show that because apatinib treatment could induce ER stress-related apoptosis and protective autophagy in human CRC cell lines, targeting autophagy is a promising therapeutic strategy to relieve apatinib drug resistance in CRC.

LIFR promotes tumor angiogenesis by up-regulating IL-8 levels in colorectal cancer.

Leukemia inhibitory factor receptor (LIFR) has been documented as a cancer promoter and to be present at high levels in various types of tumor tissues. In our search for molecules prognostic of colorectal cancer (CRC), we found high levels of LIFR in CRC tissue samples. Further analyses revealed that LIFR was indeed prognostic of CRC patient survival, and was associated with tumor size, lymphatic metastasis and stages. LIFR was found to promote tumor growth, metastasis and angiogenesis both in vitro and in vivo. High levels of LIFR in CRC facilitated proliferation and migration of endothelial cells, resulting in an increase in angiogenic activity. Moreover, interleukin 8 (IL-8) was found to play a role in the LIFR induced angiogenesis. IL-8 levels were correlated with LIFR levels in CRC tissues, whereas depletion of IL-8 led to a reduced angiogenic activity of LIFR in CRC cells. In addition, LIFR increased phosphorylation level of Erk, which regulates il-8 transcription. We conclude that LIFR is possibly a valuable prognostic marker for CRC. Our results also implicate a mechanism by which LIFR regulates tumor angiogenesis through Erk/IL-8 pathway, and that LIFR could be a potential therapeutic target for CRC.

MicroRNA-378-5p suppresses cell proliferation and induces apoptosis in colorectal cancer cells by targeting BRAF.

MicroRNAs (miRNAs) are a group of small non-coding RNA molecules that potentially play a critical role in tumorigenesis. Increasing evidences indicate that miR-378-5p is dysregulated in numerous human cancers including colorectal cancer (CRC) which hypothesizes that miR-378-5p may play an important role in tumorigenesis. However, its role in CRC carcinogenesis remains poorly defined because of lacking target genes information. In the present study, it was demonstrated that the expression of miR-378-5p was down-regulated in CRC tissues and cell lines as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Furthermore, overexpression of miR-378-5p suppressed cell proliferation, as indicated by CCK-8 assay. Flow cytometric analysis demonstrated that overexpression of miR-378-5p induced cell cycle arrest and promoted apoptosis in CRC cells. A luciferase reporter assay indicated that BRAF was a direct target of miR-378-5p. Western blot and qRT-PCR analysis indicated that BRAF was significantly down-regulated by miR-378-5p in CRC cells. Moreover, miR-378-5p was negatively associated with BRAF in CRC tissues compared to adjacent non-tumor tissues. These results demonstrate that down-regulation of miR-378-5p promotes CRC cells growth by targeting BRAF and restoration of their levels is a potentially promising therapeutic in CRC.